KRIBIOLISA™ Denosumab (PROLIA™) C1q Binding ELISA

KRIBIOLISA™ Denosumab (PROLIA™) C1q Binding ELISA

REF : KBI3026 Ver 1.0 RUO

Immunoassay for the determination of Denosumab C1q Binding.

RUO For Research Use Only REF Catalog Number

Store At LOT Batch Code

Manufactured By Biological Risk

Expiry Date Consult Operating Instructions

For Research or Manufacturing Use Only. Purchase does not include or carry the right to resell or transfer this product either as a stand-alone product or as a component of another product. Any use of this product other than the permitted use without the express written authorization of KRISHGEN BioSystems is strictly prohibited.

KRISHGEN BioSystems | Unit Nos#318/319, Shah & Nahar, Off Dr E Moses Road, Worli Mumbai 400018. India. | Tel: 91(22) 49198700 | Email: [email protected]

Cat No#KBI3026, Ver1.0 www.krishgen.com 1 KRIBIOLISA™ Denosumab (PROLIA™) C1q Binding ELISA

Introduction:

The complement component 1q (or simply C1q) is a protein complex involved in the complement system, which is part of the innate immune system. C1q together with C1r and C1s form the C1 complex. Antibodies of the adaptive immune system can bind antigen, forming an antigen-antibody complex. When C1q binds antigen-antibody complexes, the C1 complex becomes activated. Activation of the C1 complex initiates the classical complement pathway of the complement system.

Intended Use:

This KRIBIOLISA™ Denosumab C1q Binding ELISA is used as an analytical tool for determination of Denosumab C1q Binding.

Principle:

The method employs the quantitative enzyme immunoassay technique. PROLIA™ and Proposed Biosimilar Denosumab are pre-coated onto microwells. Human C1q standards or samples are added to microwells. After incubation and washing, Anti-C1q-HRP Conjugate is pipetted into microwells. An incubation followed by washing cycle, the ready to use substrate solution (TMB) is added to microwells and color develops proportionally to the amount of C1q in Standard or in sample. Color development is then stopped by addition of stop solution. Absorbance is measured at 450 nm

Materials Provided (for quantitative determination of C1q and sufficient reagents to perform 4 x 96 tests):

1. PROLIA™ Coated Microtiter Plate (12x8 wells) – 1 no 2. Uncoated Microtiter Plate (12x8 wells) – 3 no 3. Coating Buffer (2X) – 20 ml 4. Blocking Reagent (5X) – 12 ml 5. Stabilizing Reagent (1X) – 36 ml 6. C1q Standard, (0.1 ml/vial) – (0, 0.3, 0.6, 1.2, 2.4, 4.8, 9.6 and 19.2) ug/ml 7. Anti-C1q-HRP Conjugate – 25 ml 8. Assay Diluent – 110 ml 9. Wash Buffer (20X) – 120 ml 10. TMB Substrate – 45 ml 11. Stop Solution – 45 ml 12. Activated Silica Gel – 6 no 13. Polybag – 6 no 14. Aluminum Pouch – 3 no 15. Instruction Manual

Materials to be provided by the End-User:

1. Microtiter Plate Reader able to measure absorbance at 450 nm. 2. Adjustable pipettes and multichannel pipettor to measure volumes ranging from 25 ul to 1000 ul 3. Deionized (DI) water 4. Wash bottle or automated microplate washer 5. Semi-Log graph paper or software for data analysis 6. Timer 7. Absorbent Pape

Handling/Storage:

1. All reagents should be stored at 20C to 80C for stability.

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2. All the reagents and wash solutions should be used within 12 months from manufacturing date. 3. Before using, bring all components to room temperature (18-25ºC). Upon assay completion ensure all components of the kit are returned to appropriate storage conditions. 4. The Substrate is light-sensitive and should be protected from direct sunlight or UV sources.

Health Hazard Warnings:

1. Reagents that contain preservatives may be harmful if ingested, inhaled or absorbed through the skin. 2. For Research or Manufacturing use only.

Sample Preparation and Storage: Preparation before Use Allow samples to reach room temperature prior to assay. Take care to agitate patient samples gently in order to ensure homogeneity.

Coating of Biosimilar Denosumab (test sample) Microtiter Plates

DAY 1 To make Coating Buffer (1X): dilute 20 ml of Coating Buffer (2X) in 20 ml of DI water.

1. Prepare three coating solution of Biosimilar Denosumab in 10.0 ml of Coating Buffer (1X): Coating Concentration of Biosimilar Denosumab in three different solutions will be 2.8 ug/ml, 4.0 ug/ml and 5.2 ug/ml.

2. Add 100 ul of Coating Solution of Biosimilar Denosumab to Uncoated Microtiter Plate (12x8 wells) and store at 2-8°C for 16-18hr. Care should be taken that the plates are kept in a tight polybag to avoid water condensation affecting the wells.

DAY 2 Decant and tap dry onto absorbent paper. To make Blocking Reagent (1X): dilute 2 ml of Blocking Reagent (5X) in 10 ml of DI water.

1. Add 200 ul of Blocking Reagent (1X) and incubate at RT (20°C to -25°C) for 1.0hr.

2. Decant and tap dry onto absorbent paper.

3. Add 100 ul of Stabilizing Reagent (1X) and incubate at RT (20°C to -25°C) for 1.0hr.

4. Decant and tap dry onto absorbent paper.

5. Keep the coated plates for drying at 44°C ± 1°C. for 1.0hr.

6. Pack the one coated plate per aluminum pouch containing two activated silica gel.

Reagent Preparation (all reagents should be diluted immediately prior to use):

1. Label any aliquots made with the kit Lot No and Expiration date and store it at appropriate conditions mentioned.

2. Bring all reagents to Room Temperature before use.

3. To make Wash Buffer (1X); dilute 50 ml of Wash Buffer (20X) in 950 ml of DI water.

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Procedural Notes:

1. In order to achieve good assay reproducibility and sensitivity, proper washing of the plates to remove excess un-reacted reagents is essential. 2. High Dose Hook Effect may be observed in samples with very high concentrations of C1q. If Hook Effect is possible, the samples to be assayed should be diluted with a compatible diluent. Thus if the C1q concentration of the undiluted sample is less than the diluted sample, this may be indicative of the Hook Effect. 3. Avoid assay of Samples containing sodium azide (NaN3), as it could destroy the HRP activity resulting in under-estimation of the amount of C1q. 4. It is recommended that all Standards and Samples be assayed in duplicates. 5. Maintain a repetitive timing sequence from well to well for all the steps to ensure that the incubation timings are same for each well. 6. If the Substrate has a distinct blue color prior to use it may have been contaminated and use of such substrate could compromise the sensitivity of the assay. 7. The plates should be read within 30 minutes after adding the Stop Solution. 8. Make a work list in order to identify the location of Standards and Samples.

Assay Procedure:

Bring all reagents to room temperature prior to use. It is strongly recommended that all Standards and Samples be run in duplicates or triplicates. A standard curve is required for each assay

1. Dispense 150 ul of Assay Diluent into respective wells.

2. Pipette 10 ul of C1Q Standard into respective wells. Mix gently.

3. Cover the plate and Incubate at 37ºC for 2 hours.

4. Aspirate and Wash plate 5 times with Wash Buffer (1X) and blot residual buffer by firmly tapping plate upside down on absorbent paper. Wipe of any liquid from the bottom outside of the microtiter wells as any residue can interfere in the reading step.

5. Add 100 ul of Assay Diluent and 50 ul of Anti-C1q-HRP Conjugate in each well.

6. Cover the plate and Incubate at 37ºC for 30 mins.

7. Aspirate and Wash plate 5 times with Wash Buffer (1X) and blot residual buffer by firmly tapping plate upside down on absorbent paper. Wipe of any liquid from the bottom outside of the microtiter wells as any residue can interfere in the reading step.

7. Add 100 ul of TMB Substrate in each well.

8. Incubate the plate at Room Temperature for 15 minutes in dark. DO NOT SHAKE or else it may result in higher backgrounds and worse precision. Positive wells should turn bluish in color.

9. Pipette out 100 ul of Stop Solution. Wells should turn from blue to yellow in color.

10. Read the Absorbance at 450 nm with a microplate reader.

Cat No#KBI3026, Ver1.0 www.krishgen.com 4 KRIBIOLISA™ Denosumab (PROLIA™) C1q Binding ELISA

SCHEMATIC WORKING FLOW

Dispense 150 ul of Assay Diluent per well.  Dispense 10 ul of C1q Standard / Sample per well, mix gently.  Cover plate and Incubate for 2.0 hr at 37°C.  Decant, then wash each well 5 times with 300 ul wash solution (with 30s soak time) and tap dry onto absorbent paper.  Dispense 100 ul of Assay Diluent and 50 ul of Anti-C1q-HRP Conjugate per well  Cover plate and Incubate for 30 min at 37°C.  Decant, then Wash each well 5 times with 300 ul Wash Solution (with 30s soak time) and tap dry onto absorbent paper.  Dispense 100 ul TMB Substrate per well.  Incubate for 15-30 min at 37°C protected from light.  Dispense 100 ul Stop Solution per well, mix gently.  Read OD at 450 nm with a microplate reader within 30 min after reaction stop.

Typical Example of a Work list

Absorbance at Well # Contents Mean Absorbance μg/ml C1q equiv. 450nm 1A Zero Standard 2A Zero Standard 1B 0.3 μg/ml 2B 0.3 μg/ml 1C 0.6 μg/ml 2C 0.6 μg/ml 1D 1.2 μg/ml 2D 1.2 μg/ml 1E 2.4 μg/ml 2E 2.4 μg/ml 1F 4.8 μg/ml 2F 4.8 μg/ml 1G 9.6 μg/ml 2G 9.6 μg/ml 1H 19.2 μg/ml 2H 19.2 μg/ml

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Calculation of Results:

Determine the Mean Absorbance for each set of duplicate or triplicate Standards and Samples. Using Semi- Log graph paper, plot the average value (absorbance 450nm) of each standard on the Y-axis versus the corresponding concentration of the standards on the X-axis. Draw the best fit curve through the standard points. To determine the unknown C1q concentrations, find the unknown’s Mean Absorbance value on the Y- axis and draw a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the X- axis and read the C1q Concentration. If samples were diluted, multiply by the appropriate dilution factor. Software which is able to generate a cubic spline curve-fit is best recommended for automated results.

1. Criteria used:

Qualification Principle Qualification Parameters Acceptance Criteria Assay Linearity & Range Correlation coefficient > 0.95 Y-intercept - 15% - 15% Slope 0.8 - 1.2 Residual sum of squares Report Results Range Range Report Results Repeatability %CV Mean Range (Intra-Assay Precision) Intermediate Precision %CV Mean Range (Inter-Assay Precision) Accuracy Recovery 80% - 120%

2. Calculate the EC50 for each line, and report their ratio (relative binding) as per regulatory requirements including using parallel line assay. For more information and options on data reduction for current regulatory requirements and for reportable values please contact us at [email protected].

Note: It is recommended to repeat the assay at a different dilution factor in the following cases: - If the sample absorbance value is below the first standard. - If the absorbance value is equivalent or higher than the 19.2 μg/ml standard.

Quality Control:

It is recommended that for each laboratory assay appropriate quality control samples in each run to be used to ensure that all reagents and procedures are correct.

Safety Precautions:

• This kit is for in vitro use only. Follow the working instructions carefully. • The expiration dates stated on the kit are to be observed. The same relates to the stability stated for reagents • Do not use or mix reagents from different lots. • Do not use reagents from other manufacturers. • Avoid time shift during pipetting of reagents. • All reagents should be kept in the original shipping container. • Some of the reagents contain small amount of sodium azide (< 0.1 % w/w) as preservative. They must not be swallowed or allowed to come into contact with skin or mucosa. • Source materials maybe derived from human body fluids or organs used in the preparation of this kit were tested and found negative for HBsAg and HIV as well as for HCV antibodies. However, no known test guarantees the absence of such viral agents. Therefore, handle all components and all patient samples as if potentially hazardous.

Cat No#KBI3026, Ver1.0 www.krishgen.com 6 KRIBIOLISA™ Denosumab (PROLIA™) C1q Binding ELISA

• Since the kit contains potentially hazardous materials, the following precautions should be observed - Do not smoke, eat or drink while handling kit material - Always use protective gloves - Never pipette material by mouth - Wipe up spills promptly, washing the affected surface thoroughly with a decontaminant. • In any case GLP should be applied with all general and individual regulations to the use of this kit.

REFERENCES 1. European Medicines Agency. Similar biological medicinal products containing monoclonal antibodies: non-clinical and clinical issues. EMA/CHMP/BMWP/403543/2010. Jun 2012. 2. Revision of the guideline on immunogenicity assessment of biotechnology-derived therapeutic proteins. EMA/275542/2013 Mar 2014. 3. United States FDA. Guidance for industry: Scientific Considerations in Demonstrating Biosimilarity to a Reference Product. Feb 2012. 4. United States FDA. Guidance for Industry: Quality Considerations in Demonstrating Biosimilarity to a Reference Protein Product. Feb 2012. 5. United States FDA. Guidance for Industry: Clinical Pharmacology Data to Support a Demonstration of Biosimilarity to a Reference Product. May 2014 6. Similar biological medicinal products containing biotechnology-derived proteins as active substance: non-clinical and clinical issues. EMEA/CHMP/BMWP/42832/2005 Feb 2006. EMEA/CHMP/BMWP/ 42832/2005 Rev. 1 Jun 2013.

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Krishgen Biosystems does not warrant against damages or defects arising in shipping or handling, or out of accident or improper or abnormal use of the Products; against defects in products or components not manufactured by Krishgen Biosystems, or against damages resulting from such non-Krishgen Biosystems made products or components. Krishgen Biosystems passes on to customer the warranty it received (if any) from the maker thereof of such non Krishgen made products or components. This warranty also does not apply to Products to which changes or modifications have been made or attempted by persons other than pursuant to written authorization by Krishgen Biosystems.

THIS WARRANTY IS EXCLUSIVE. The sole and exclusive obligation of Krishgen Biosystems shall be to repair or replace the defective Products in the manner and for the period provided above. Krishgen Biosystems shall not have any other obligation with respect to the Products or any part thereof, whether based on contract, tort, and strict liability or otherwise. Under no circumstances, whether based on this Limited Warranty or otherwise, shall Krishgen Biosystems be liable for incidental, special, or consequential damages.

This Limited Warranty states the entire obligation of Krishgen Biosystems with respect to the Products. If any part of this Limited Warranty is determined to be void or illegal, the remainder shall remain in full force and effect.

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Trademarks. KRIBIOLISA™ and KRISHGEN BIOSYSTEMS are registered trademarks of KRISHGEN BIOYSTEMS. PROLIA™ is a registered trademark of Amgen, Inc. The use of the mark is only indicative and does not in any way construe authorization or usage right.

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